Tenasin-C nucleic acid ligands

ABSTRACT

Methods are described for the identification and preparation of nucleic acid ligands to tenascin-C. Included in the invention are specific RNA ligands to tenascin-C identified by the SELEX method. Further included in the invention are methods for detecting the presence of a disease condition in a biological tissue in which tenascin-C is expressed.

RELATED APPLICATIONS

[0001] This application is a Divisional of U.S. patent application Ser.No. 09/854,662, filed May 14, 2001, which is a Divisional of U.S. patentapplication Ser. No. 09/364,902, filed Jul. 29, 1999, now U.S. Pat. No.6,232,071, which is a Continuation-in-Part of U.S. patent applicationSer. No. 08/434,425, filed May 3, 1995, entitled Systematic Evolution ofLigands by Exponential Enrichment: Tissue SELEX,” now U.S. Pat. No.5,789,157, which is a Continuation-in Part of U.S. patent applicationSer. No. 07/714,131, filed Jun. 10, 1991, entitled “Nucleic AcidLigands,” now U.S. Pat. No. 5,475,096, which is a Continuation-in-Partof U.S. patent application Ser. No. 07/536,428, filed Jun. 11, 1990,entitled “Systematic Evolution of Ligands by Exponential Enrichment,”now abandoned, and U.S. patent application Ser. No. 07/964,624, filedOct. 21, 1992, entitled “Nucleic Acid Ligands to HIV-RT and HIV-1 Rev”,now U.S. Pat. No. 5,496,938. This application is also aContinuation-in-Part of U.S. patent application Ser. No. 08/993,765,filed Dec. 18, 1997, entitled “Nucleotide Based Prodrugs.” Each of theabove described patents and applications is specifically incorporated byreference herein in their entirety.

FIELD OF THE INVENTION

[0002] Described herein are high affinity nucleic acid ligands totenascin-C. Also described herein are methods for identifying andpreparing high affinity nucleic acid ligands to tenascin-C. The methodused herein for identifying such nucleic acid ligands is called SELEX,an acronym for Systematic Evolution of Ligands by Exponentialenrichment. Further disclosed are high affinity nucleic acid ligands totenascin-C. Further disclosed are RNA ligands to tenascin-C. Alsoincluded are oligonucleotides containing nucleotide derivativeschemically modified at the 2′-positions of the purines and pyrimidines.Additionally disclosed are RNA ligands to tenascin-C containing 2′-F and2′OMe modifications. The oligonucleotides of the present invention areuseful as diagnostic and/or therapeutic agents.

BACKGROUND OF THE INVENTION

[0003] Tenascin-C is a 1.1-1.5 million Da, hexameric glycoprotein thatis located primarily in the extracellular matrix. Tenascin-C isexpressed during embryogenesis, wound healing, and neoplasia, suggestinga role for this protein in tissue remodeling (Erickson & Bourdon, (1989)Ann Rev Cell Biol 5:71-92). Neoplastic processes also involve tissueremodeling, and tenascin-C is over-expressed in many tumor typesincluding carcinomas of the lung, breast, prostate, and colon,astrocytomas, glioblastomas, melanomas, and sarcomas (Soini et al.,(1993) Am J Clin Pathol 100(2):145-50; Koukoulis et al., (1991) HumPathol 22(7):636-43; Borsi et al., (1992) Int J Cancer 52(5):688-92;Koukoulis et al., (1993) J Submicrosc Cytol Pathol 25(2):285-95; Ibrahimet al., (1993) Hum Pathol 24(9):982-9; Riedl et al., (1998) Dis ColonRectum 41(1):86-92; Tuominen & Kallioinen (1994) J Cutan Pathol21(5):424-9; Natali et al., (1990) Int J Cancer 46(4):586-90; Zagzag etal., (1995) Cancer Res 55(4):907-14; Hasegawa et al., (1997) ActaNeuropathol (Berl) 93(5):431-7; Saxon et al., (1997) Pediatr Pathol LabMed 17(2):259-66; Hasegawa et al., (1995) Hum Pathol 26(8):838-45). Inaddition, tenascin-C is overexpressed in hyperproliferative skindiseases, e.g. psoriasis (Schalkwijk et al., (1991) Br J Dermatol124(1):13-20), and in atherosclerotic lesions (Fukumoto et al., (1998) JAtheroscler Thromb 5(1):29-35; Wallner et al., (1999) Circulation99(10):1284-9). Radiolabeled antibodies that bind tenascin-C are usedfor imaging and therapy of tumors in clinical settings (Paganelli etal., (1999) Eur J Nucl Med 26(4):348-57; Paganelli et al., (1994) Eur JNucl Med 21(4):314-21; Bigner et al., (1998) J Clin Oncol 16(6):2202-12;Merlo et al., (1997) Int J Cancer 71(5):810-6).

[0004] Aptamers against tenascin-C have potential utility for cancerdiagnosis and therapy, as well as for diagnosis and therapy ofatheroslerosis and therapy of psoriasis. Relative to antibodies,aptamers are small (7-20 kDa), clear very rapidly from blood, and arechemically synthesized. Rapid blood clearance is important for in vivodiagnostic imaging, where blood levels are a primary determinant ofbackground that obscures an image. Rapid blood clearance may also beimportant in therapy, where blood levels may contribute to toxicity.SELEX technology allows rapid aptamer isolation, and chemical synthesisenables facile and site-specific conjugation of aptamers to a variety ofinert and bioactive molecules. An aptamer to tenascin-C would thereforebe useful for tumor therapy or in vivo or ex vivo diagnostic imagingand/or for delivering a variety of therapeutic agents complexed with thetenascin-C nucleic acid ligand for treatment of disease conditions inwhich tenascin-C is expressed.

[0005] The dogma for many years was that nucleic acids had primarily aninformational role. Through a method known as Systematic Evolution ofLigands by EXponential enrichment, termed the SELEX process, it hasbecome clear that nucleic acids have three dimensional structuraldiversity not unlike proteins. The SELEX process is a method for the invitro evolution of nucleic acid molecules with highly specific bindingto target molecules and is described in U.S. patent application Ser. No.07/536,428, filed Jun. 11, 1990, entitled “Systematic Evolution ofLigands by EXponential Enrichment,” now abandoned, U.S. Pat. No.5,475,096 entitled “Methods for Identifying Nucleic Acid Ligands”, U.S.Pat. No. 5,270,163 (see also WO 91/19813) entitled “Nucleic AcidLigands” each of which is specifically incorporated by reference hereinin its entirety. Each of these applications, collectively referred toherein as the SELEX Patent Applications, describes a fundamentally novelmethod for making a nucleic acid ligand to any desired target molecule.The SELEX process provides a class of products which are referred to asnucleic acid ligands or aptamers, each having a unique sequence, andwhich have the property of binding specifically to a desired targetcompound or molecule. Each SELEX-identified nucleic acid ligand is aspecific ligand of a given target compound or molecule. The SELEXprocess is based on the unique insight that nucleic acids havesufficient capacity for forming a variety of two- and three-dimensionalstructures and sufficient chemical versatility available within theirmonomers to act as ligands (form specific binding pairs) with virtuallyany chemical compound, whether monomeric or polymeric. Molecules of anysize or composition can serve as targets in the SELEX method. The SELEXmethod applied to the application of high affinity binding involvesselection from a mixture of candidate oligonucleotides and step-wiseiterations of binding, partitioning and amplification, using the samegeneral selection scheme, to achieve virtually any desired criterion ofbinding affinity and selectivity. Starting from a mixture of nucleicacids, preferably comprising a segment of randomized sequence, the SELEXmethod includes steps of contacting the mixture with the target underconditions favorable for binding, partitioning unbound nucleic acidsfrom those nucleic acids which have bound specifically to targetmolecules, dissociating the nucleic acid-target complexes, amplifyingthe nucleic acids dissociated from the nucleic acid-target complexes toyield a ligand-enriched mixture of nucleic acids, then reiterating thesteps of binding, partitioning, dissociating and amplifying through asmany cycles as desired to yield highly specific high affinity nucleicacid ligands to the target molecule.

[0006] It has been recognized by the present inventors that the SELEXmethod demonstrates that nucleic acids as chemical compounds can form awide array of shapes, sizes and configurations, and are capable of a farbroader repertoire of binding and other functions than those displayedby nucleic acids in biological systems.

[0007] The basic SELEX method has been modified to achieve a number ofspecific objectives. For example, U.S. patent application Ser. No.07/960,093, filed Oct. 14, 1992, now abandoned, and U.S. Pat. No.5,707,796, both entitled “Method for Selecting Nucleic Acids on theBasis of Structure,” describe the use of the SELEX process inconjunction with gel electrophoresis to select nucleic acid moleculeswith specific structural characteristics, such as bent DNA. U.S. patentapplication Ser. No. 08/123,935, filed Sep. 17, 1993, entitled“Photoselection of Nucleic Acid Ligands,” now abandoned, U.S. Pat. No.5,763,177 entitled “Systematic Evolution of Ligands by ExponentialEnrichment: Photoselection of Nucleic Acid Ligands and Solution SELEX”and U.S. patent application Ser. No. 09/093,293, filed Jun. 8 1998,entitled “Systematic Evolution of Ligands by Exponential Enrichment:Photoselection of Nucleic Acid Ligands and Solution SELEX” describe aSELEX based method for selecting nucleic acid ligands containingphotoreactive groups capable of binding and/or photocrosslinking toand/or photoinactivating a target molecule. U.S. Pat. No. 5,580,737entitled “High-Affinity Nucleic Acid Ligands That Discriminate BetweenTheophylline and Caffeine,” describes a method for identifying highlyspecific nucleic acid ligands able to discriminate between closelyrelated molecules, which can be non-peptidic, termed Counter-SELEX. U.S.Pat. No. 5,567,588 entitled “Systematic Evolution of Ligands byEXponential Enrichment: Solution SELEX,” describes a SELEX-based methodwhich achieves highly efficient partitioning between oligonucleotideshaving high and low affinity for a target molecule.

[0008] The SELEX method encompasses the identification of high-affinitynucleic acid ligands containing modified nucleotides conferring improvedcharacteristics on the ligand, such as improved in vivo stability orimproved delivery characteristics. Examples of such modificationsinclude chemical substitutions at the ribose and/or phosphate and/orbase positions. SELEX process-identified nucleic acid ligands containingmodified nucleotides are described in U.S. Pat. No. 5,660,985 entitled“High Affinity Nucleic Acid Ligands Containing Modified Nucleotides,”that describes oligonucleotides containing nucleotide derivativeschemically modified at the 5- and 2′-positions of pyrimidines. U.S. Pat.No. 5,580,737, supra, describes highly specific nucleic acid ligandscontaining one or more nucleotides modified with 2′-amino (2′-NH₂),2′-fluoro (2′-F), and/or 2′-O-methyl (2′-OMe). U.S. patent applicationSer. No. 08/264,029, filed Jun. 22, 1994, entitled “Novel Method ofPreparation of Known and Novel 2′ Modified Nucleosides by IntramolecularNucleophilic Displacement,” now abandoned, oligonucleotides containingvarious 2′-modified pyrimidines.

[0009] The SELEX method encompasses combining selected oligonucleotideswith other selected oligonucleotides and non-oligonucleotide functionalunits as described in U.S. Pat. No. 5,637,459 entitled “SystematicEvolution of Ligands by EXponential Enrichment: Chimeric SELEX,” andU.S. Pat. No. 5,683,867 entitled “Systematic Evolution of Ligands byEXponential Enrichment: Blended SELEX,” respectively. These applicationsallow the combination of the broad array of shapes and other properties,and the efficient amplification and replication properties, ofoligonucleotides with the desirable properties of other molecules.

[0010] The SELEX method further encompasses combining selected nucleicacid ligands with lipophilic compounds or non-immunogenic, highmolecular weight compounds in a diagnostic or therapeutic complex asdescribed in U.S. patent application Ser. No. 08/434,465, filed May 4,1995, entitled “Nucleic Acid Ligand Complexes”. Each of the abovedescribed patents and applications which describe modifications of thebasic SELEX procedure are specifically incorporated by reference hereinin their entirety.

SUMMARY OF THE INVENTION

[0011] The present invention describes a method for isolating nucleicacid ligands that bind to tenascin-C with high specificity. Furtherdescribed herein are nucleic acid ligands to tenascin-C. Also describedherein are high affinity RNA ligands to tenascin-C. Further describedare 2′fluoro-modified pyrimidine and 2′OMe-modified purine RNA ligandsto tenascin-C. The method utilized herein for identifying such nucleicacid ligands is called SELEX, an acronym for Systematic Evolution ofLigands by Exponential enrichment. Included herein are the ligands thatare shown in Tables 3 and 4 and FIG. 2.

[0012] Further included in this invention is a method for detecting thepresence of a disease that is expressing tenascin-C in a biologicaltissue that may contain the disease. Still further included in thisinvention is a method for detecting the presence of a tumor that isexpressing tenascin-C in a biological tissue that may contain the tumor.Further included in this invention is a complex for use in in vivo or exvivo diagnostics. Still further included in this invention is a methodfor delivering therapeutic agents for the treatment or prophylaxis ofdiseased tissues that express tenascin-C. Still further included in thisinvention is a complex for use in delivering therapeutic agents fortreatment or prophylaxis of diseased tissues that express tenascin-C.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013]FIG. 1 shows binding of Cell SELEX RNA pools to U251 cells.

[0014]FIG. 2 shows proposed secondary structure of aptamers TTA1 andTTA1.NB. Included in the figure is the conjugation of the aptamers withTc-99m. All A's are 2′OMe modified. All G's, except as indicated, are2′OMe modified. All C's and U's are 2′F modified.

[0015]FIG. 3 shows images of U251 tumor xenografts in mice, obtainedusing Tc-99m-labeled TTA1 and TTA1.NB, three hours post-injection.

[0016]FIG. 4 shows fluorescence microscopy of a U251 glioblastoma tumorsection, taken three hours after i.v. injection ofRhodamine-Red-X-labeled TTA1.

[0017]FIG. 5 shows the way in which the Tc-99m and linker is boundthrough the 5′G of TTA1.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] The central method utilized herein for identifying nucleic acidligands to tenascin-C is called the SELEX process, an acronym forSystematic Evolution of Ligands by Exponential enrichment and involves(a) contacting the candidate mixture of nucleic acids with tenascin-C(b) partitioning between members of said candidate mixture on the basisof affinity to tenascin-C, and c) amplifying the selected molecules toyield a mixture of nucleic acids enriched for nucleic acid sequenceswith a relatively higher affinity for binding to tenascin-C. Theinvention includes RNA ligands to tenascin-C. This invention furtherincludes the specific RNA ligands to tenascin-C shown in Tables 3 and 4and FIG. 2. More specifically, this invention includes nucleic acidsequences that are substantially homologous to and that havesubstantially the same ability to bind tenascin-C as the specificnucleic acid ligands shown in Tables 3 and 4 and FIG. 2. Bysubstantially homologous it is meant a degree of primary sequencehomology in excess of 70% , most preferably in excess of 80%, and evenmore preferably in excess of 90%, 95%, or 99%. The percentage ofhomology as described herein is calculated as the percentage ofnucleotides found in the smaller of the two sequences which align withidentical nucleotide residues in the sequence being compared when 1 gapin a length of 10 nucleotides may be introduced to assist in thatalignment. Substantially the same ability to bind tenascin-C means thatthe affinity is within one or two orders of magnitude of the affinity ofthe ligands described herein. It is well within the skill of those ofordinary skill in the art to determine whether a givensequence—substantially homologous to those specifically describedherein—has the same ability to bind tenascin-C.

[0019] A review of the sequence homologies of the nucleic acid ligandsof tenascin-C shown in Tables 3 and 4 and FIG. 2 shows that sequenceswith little or no primary homology may have substantially the sameability to bind tenascin-C. For these reasons, this invention alsoincludes Nucleic Acid Ligands that have substantially the samepostulated structure or structural motifs and ability to bind tenascin-Cas the nucleic acid ligands shown in Tables 3 and 4 and FIG. 2.Substantially the same structure or structural motifs can be postulatedby sequence alignment using the Zukerfold program (see Zuker (1989)Science 244:48-52). As would be known in the art, other computerprograms can be used for predicting secondary structure and structuralmotifs. Substantially the same structure or structural motif of NucleicAcid Ligands in solution or as a bound structure can also be postulatedusing NMR or other techniques as would be known in the art.

[0020] Further included in this invention is a method for detecting thepresence of a disease that is expressing tenascin-C in a biologicaltissue which may contain the disease by the method of (a) identifying anucleic acid ligand from a candidate mixture of nucleic acids, thenucleic acid ligand being a ligand of tenascin-C, by the methodcomprising (i) contacting a candidate mixture of nucleic acids withtenascin-C, wherein nucleic acids having an increased affinity totenascin-C relative to the candidate mixture may be partitioned from theremainder of the candidate mixture; (ii) partitioning the increasedaffinity nucleic acids from the remainder of the candidate mixture;(iii) amplifying the increased affinity nucleic acids to yield a mixtureof nucleic acids with relatively higher affinity and specificity forbinding to tenascin-C, whereby a nucleic acid ligand of tenascin-C isidentified; (b) attaching a marker that can be used in in vivo or exvivo diagnostics to the nucleic acid ligand identified in step (iii) toform a marker-nucleic acid ligand complex; (c) exposing a tissue whichmay contain the disease to the marker-nucleic acid ligand complex; and(d) detecting the presence of the marker-nucleic acid ligand in thetissue, whereby a disease expressing tenascin-C is identified.

[0021] It is a further object of the present invention to provide acomplex for use in in vivo or ex vivo diagnostics comprising one or moretenascin-C nucleic acid ligands and one or more markers. Still furtherincluded in this invention is a method for delivering therapeutic agentsfor the treatment or prophylaxis of disease conditions in whichtenascin-C is expressed. Still further included in this invention is acomplex for use in delivering therapeutic agents for treatment orprophylaxis of disease conditions in which tenascin-C is expressed.

Definitions

[0022] Various terms are used herein to refer to aspects of the presentinvention. To aid in the clarification of the description of thecomponents of this invention, the following definitions are provided:

[0023] As used herein, “nucleic acid ligand” is a non-naturallyoccurring nucleic acid having a desirable action on a target. Nucleicacid ligands are often referred to as “aptamers.” The target of thepresent invention is tenascin-C, hence the term tenascin-C nucleic acidligand. A desirable action includes, but is not limited to, binding ofthe target, catalytically changing the target, reacting with the targetin a way which modifies/alters the target or the functional activity ofthe target, covalently attaching to the target as in a suicideinhibitor, facilitating the reaction between the target and anothermolecule. In the preferred embodiment, the action is specific bindingaffinity for a target molecule, such target molecule being a threedimensional chemical structure other than a polynucleotide that binds tothe nucleic acid ligand through a mechanism which predominantly dependson Watson/Crick base pairing or triple helix binding, wherein thenucleic acid ligand is not a nucleic acid having the known physiologicalfunction of being bound by the target molecule. Nucleic acid ligands areidentified from a candidate mixture of nucleic acids, said nucleic acidligand being a ligand of a tenascin-C, by the method comprising: a)contacting the candidate mixture with tenascin-C, wherein nucleic acidshaving an increased affinity to tenascin-C relative to the candidatemixture may be partitioned from the remainder of the candidate mixture;b) partitioning the increased affinity nucleic acids from the remainderof the candidate mixture; and c) amplifying the increased affinitynucleic acids to yield a ligand-enriched mixture of nucleic acids (seeU.S. patent application Ser. No. 08/434,425, filed May 3, 1995, now U.S.Pat. No. 5,789,157, which is hereby incorporated herein by reference).

[0024] As used herein, “candidate mixture” is a mixture of nucleic acidsof differing sequence from which to select a desired ligand. The sourceof a candidate mixture can be from naturally-occurring nucleic acids orfragments thereof, chemically synthesized nucleic acids, enzymaticallysynthesized nucleic acids or nucleic acids made by a combination of theforegoing techniques. In a preferred embodiment, each nucleic acid hasfixed sequences surrounding a randomized region to facilitate theamplification process.

[0025] As used herein, “nucleic acid” means either DNA, RNA,single-stranded or double-stranded, and any chemical modificationsthereof. Modifications include, but are not limited to, those whichprovide other chemical groups that incorporate additional charge,polarizability, hydrogen bonding, electrostatic interaction, andfluxionality to the nucleic acid ligand bases or to the nucleic acidligand as a whole. Such modifications include, but are not limited to,2′-position sugar modifications, 5-position pyrimidine modifications,8-position purine modifications, modifications at exocyclic amines,substitution of 4-thiouridine, substitution of 5-bromo or 5-iodo-uracil;backbone modifications, methylations, unusual base-pairing combinationssuch as the isobases isocytidine and isoguanidine and the like.Modifications can also include 3′ and 5′ modifications such as capping.

[0026] “SELEX” methodology involves the combination of selection ofnucleic acid ligands which interact with a target in a desirable manner,for example binding to a protein, with amplification of those selectednucleic acids. Optional iterative cycling of the selection/amplificationsteps allows selection of one or a small number of nucleic acids whichinteract most strongly with the target from a pool which contains a verylarge number of nucleic acids. Cycling of the selection/amplificationprocedure is continued until a selected goal is achieved. In the presentinvention, the SELEX methodology is employed to obtain nucleic acidligands to tenascin-C.

[0027] The SELEX methodology is described in the SELEX PatentApplications.

[0028] “SELEX target” or “target” means any compound or molecule ofinterest for which a ligand is desired. A target can be a protein,peptide, carbohydrate, polysaccharide, glycoprotein, hormone, receptor,antigen, antibody, virus, substrate, metabolite, transition stateanalog, cofactor, inhibitor, drug, dye, nutrient, growth factor, etc.without limitation. In this application, the SELEX target is tenascin-C.

[0029] “Complex” as used herein means the molecular entity formed by thecovalent linking of one or more tenascin-C nucleic acid ligands with oneor more markers. In certain embodiments of the present invention, thecomplex is depicted as A-B-Y, wherein A is a marker; B is optional, andcomprises a linker; and Y is a tenascin-C nucleic acid ligand.

[0030] “Marker” as used herein is a molecular entity or entities thatwhen complexed with the tenascin-C nucleic acid ligand, either directlyor through a linker(s) or spacer(s), allows the detection of the complexin an in vivo or ex vivo setting through visual or chemical means.Examples of markers include, but are not limited to radionuclides,including Tc-99m, Re-188, Cu-64, Cu-67, F-18, ¹²⁵I, ¹³¹I, ³²P, ¹⁸⁶Re;all fluorophores, including fluorescein, rhodamine, Texas Red;derivatives of the above fluorophores, including Rhodamine-Red-X;magnetic compounds; and biotin.

[0031] As used herein, “linker” is a molecular entity that connects twoor more molecular entities through covalent bond or non-covalentinteractions, and can allow spatial separation of the molecular entitiesin a manner that preserves the functional properties of one or more ofthe molecular entities. A linker can also be known as a spacer. Examplesof a linker include, but are not limited to, the (CH₂CH₂O)₆ andhexylamine structures shown in FIG. 2.

[0032] “Therapeutic” as used herein, includes treatment and/orprophylaxis. When used, therapeutic refers to humans and other animals.

[0033] “Covalent Bond” is the chemical bond formed by the sharing ofelectrons.

[0034] “Non-covalent interactions” are means by which molecular entitiesare held together by interactions other than Covalent Bonds includingionic interactions and hydrogen bonds.

[0035] In the preferred embodiment, the nucleic acid ligands of thepresent invention are derived from the SELEX methodology. The SELEXprocess is described in U.S. patent application Ser. No. 07/536,428,entitled Systematic Evolution of Ligands by Exponential Enrichment, nowabandoned, U.S. Pat. No. 5,475,096 entitled Nucleic Acid Ligands andU.S. Pat. No. 5,270,163 (see also WO 91/19813) entitled Methods forIdentifying Nucleic Acid Ligands. These applications, each specificallyincorporated herein by reference, are collectively called the SELEXPatent Applications.

[0036] The SELEX process provides a class of products which are nucleicacid molecules, each having a unique sequence, and each of which has theproperty of binding specifically to a desired target compound ormolecule. Target molecules are preferably proteins, but can also includeamong others carbohydrates, peptidoglycans and a variety of smallmolecules. SELEX methodology can also be used to target biologicalstructures, such as cell surfaces or viruses, through specificinteraction with a molecule that is an integral part of that biologicalstructure.

[0037] In its most basic form, the SELEX process may be defined by thefollowing series of steps:

[0038] 1) A candidate mixture of nucleic acids of differing sequence isprepared. The candidate mixture generally includes regions of fixedsequences (i.e., each of the members of the candidate mixture containsthe same sequences in the same location) and regions of randomizedsequences. The fixed sequence regions are selected either: (a) to assistin the amplification steps described below, (b) to mimic a sequenceknown to bind to the target, or (c) to enhance the concentration of agiven structural arrangement of the nucleic acids in the candidatemixture. The randomized sequences can be totally randomized (i.e., theprobability of finding a base at any position being one in four) or onlypartially randomized (e.g., the probability of finding a base at anylocation can be selected at any level between 0 and 100 percent).

[0039] 2) The candidate mixture is contacted with the selected targetunder conditions favorable for binding between the target and members ofthe candidate mixture. Under these circumstances, the interactionbetween the target and the nucleic acids of the candidate mixture can beconsidered as forming nucleic acid-target pairs between the target andthose nucleic acids having the strongest affinity for the target.

[0040] 3) The nucleic acids with the highest affinity for the target arepartitioned from those nucleic acids with lesser affinity to the target.Because only an extremely small number of sequences (and possibly onlyone molecule of nucleic acid) corresponding to the highest affinitynucleic acids exist in the candidate mixture, it is generally desirableto set the partitioning criteria so that a significant amount of thenucleic acids in the candidate mixture (approximately 5-50%) areretained during partitioning.

[0041] 4) Those nucleic acids selected during partitioning as having therelatively higher affinity for the target are then amplified to create anew candidate mixture that is enriched in nucleic acids having arelatively higher affinity for the target.

[0042] 5) By repeating the partitioning and amplifying steps above, thenewly formed candidate mixture contains fewer and fewer uniquesequences, and the average degree of affinity of the nucleic acids tothe target will generally increase. Taken to its extreme, the SELEXprocess will yield a candidate mixture containing one or a small numberof unique nucleic acids representing those nucleic acids from theoriginal candidate mixture having the highest affinity to the targetmolecule.

[0043] The basic SELEX method has been modified to achieve a number ofspecific objectives. For example, U.S. patent application Ser. No.07/960,093, filed Oct. 14, 1992, now abandoned, and U.S. Pat. No.5,707,796 both entitled “Method for Selecting Nucleic Acids on the Basisof Structure,” describe the use of the SELEX process in conjunction withgel electrophoresis to select nucleic acid molecules with specificstructural characteristics, such as bent DNA. U.S. patent applicationSer. No. 08/123,935, filed Sep. 17, 1993, entitled “Photoselection ofNucleic Acid Ligands,” now abandoned, U.S. Pat. No. 5,763,177 entitled“Systematic Evolution of Ligands by Exponential Enrichment:Photoselection of Nucleic Acid Ligands and Solution SELEX” and U.S.patent application Ser. No. 09/093,293, filed Jun. 8 1998, entitled“Systematic Evolution of Ligands by Exponential Enrichment:Photoselection of Nucleic Acid Ligands and Solution SELEX” all describea SELEX based method for selecting nucleic acid ligands containingphotoreactive groups capable of binding and/or photocrosslinking toand/or photoinactivating a target molecule. U.S. Pat. No. 5,580,737entitled “High-Affinity Nucleic Acid Ligands That Discriminate BetweenTheophylline and Caffeine,” describes a method for identifying highlyspecific nucleic acid ligands able to discriminate between closelyrelated molecules, termed Counter-SELEX. U.S. Pat. No. 5,567,588entitled “Systematic Evolution of Ligands by Exponential Enrichment:Solution SELEX,” describes a SELEX-based method which achieves highlyefficient partitioning between oligonucleotides having high and lowaffinity for a target molecule. U.S. Pat. No. 5,496,938 entitled“Nucleic Acid Ligands to HIV-RT and HIV-1 Rev,” describes methods forobtaining improved nucleic acid ligands after SELEX has been performed.U.S. Pat. No. 5,705,337 entitled “Systematic Evolution of Ligands byExponential Enrichment: Chemi-SELEX,” describes methods for covalentlylinking a ligand to its target.

[0044] The SELEX method encompasses the identification of high-affinitynucleic acid ligands containing modified nucleotides conferring improvedcharacteristics on the ligand, such as improved in vivo stability orimproved delivery characteristics. Examples of such modificationsinclude chemical substitutions at the ribose and/or phosphate and/orbase positions. SELEX-identified nucleic acid ligands containingmodified nucleotides are described in U.S. Pat. No. 5,660,985 entitled“High Affinity Nucleic Acid Ligands Containing Modified Nucleotides,”that describes oligonucleotides containing nucleotide derivativeschemically modified at the 5- and 2′-positions of pyrimidines. U.S. Pat.No. 5,637,459, supra, describes highly specific nucleic acid ligandscontaining one or more nucleotides modified with 2′-amino (2′-NH₂),2′-fluoro (2′-F), and/or 2′-O-methyl (2′-OMe). U.S. patent applicationSer. No. 08/264,029, filed Jun. 22, 1994, entitled “Novel Method ofPreparation of Known and Novel 2′ Modified Nucleosides by IntramolecularNucleophilic Displacement,” describes oligonucleotides containingvarious 2′-modified pyrimidines.

[0045] The SELEX method encompasses combining selected oligonucleotideswith other selected oligonucleotides and non-oligonucleotide functionalunits as described in U.S. Pat. No. 5,637,459 entitled “SystematicEvolution of Ligands by Exponential Enrichment: Chimeric SELEX,” andU.S. Pat. No. 5,683,867 entitled “Systematic Evolution of Ligands byExponential Enrichment: Blended SELEX,” respectively. These applicationsallow the combination of the broad array of shapes and other properties,and the efficient amplification and replication properties, ofoligonucleotides with the desirable properties of other molecules.

[0046] In U.S. Pat. No. 5,496,938 methods are described for obtainingimproved Nucleic Acid Ligands after the SELEX process has beenperformed. This patent, entitled “Nucleic Acid Ligands to HIV-RT andHIV-1 Rev,” is specifically incorporated herein by reference.

[0047] U.S. patent application Ser. No. 08/434,425, entitled “SystematicEvolution of Ligands by Exponential Enrichment: Tissue SELEX,” filed May3, 1995, now U.S. Pat. No. 5,789,157, describes methods for identifyinga nucleic acid ligands to a macromolecular component of a tissue,including cancer cells, and the nucleic acid ligands so identified. Thispatent is specifically incorporated herein by reference.

[0048] One potential problem encountered in the diagnostic ortherapeutic use of nucleic acids is that oligonucleotides in theirphosphodiester form may be quickly degraded in body fluids byintracellular and extracellular enzymes such as endonucleases andexonucleases before the desired effect is manifest. Certain chemicalmodifications of the nucleic acid ligand can be made to increase the invivo stability of the nucleic acid ligand or to enhance or to mediatethe delivery of the nucleic acid ligand. See, e.g., U.S. patentapplication Ser. No. 08/117,991, filed Sep. 8, 1993, now abandoned, andU.S. Pat. No. 5,660,985, both entitled “High Affinity Nucleic AcidLigands Containing Modified Nucleotides”, which is specificallyincorporated herein by reference. Modifications of the nucleic acidligands contemplated in this invention include, but are not limited to,those which provide other chemical groups that incorporate additionalcharge, polarizability, hydrophobicity, hydrogen bonding, electrostaticinteraction, and fluxionality to the nucleic acid ligand bases or to thenucleic acid ligand as a whole. Such modifications include, but are notlimited to, 2′-position sugar modifications, 5-position pyrimidinemodifications, 8-position purine modifications, modifications atexocyclic amines, substitution of 4-thiouridine, substitution of 5-bromoor 5-iodo-uracil; backbone modifications, phosphorothioate or alkylphosphate modifications, methylations, unusual base-pairing combinationssuch as the isobases isocytidine and isoguanidine and the like.Modifications can also include 3′ and 5′ modifications such as capping.In preferred embodiments of the instant invention, the nucleic acidligands are RNA molecules that are 2′-fluoro (2′-F) modified on thesugar moiety of pyrimidine residues.

[0049] The modifications can be pre- or post-SELEX processmodifications. Pre-SELEX process modifications yield nucleic acidligands with both specificity for their SELEX target and improved invivo stability. Post-SELEX process modifications made to 2′-OH nucleicacid ligands can result in improved in vivo stability without adverselyaffecting the binding capacity of the nucleic acid ligand.

[0050] Other modifications are known to one of ordinary skill in theart. Such modifications may be made post-SELEX process (modification ofpreviously identified unmodified ligands) or by incorporation into theSELEX process.

[0051] The nucleic acid ligands of the invention are prepared throughthe SELEX methodology that is outlined above and thoroughly enabled inthe SELEX applications incorporated herein by reference in theirentirety.

[0052] The tenascin-C aptamers of the invention bind to the heparinbinding site of the tenascin-C COOH terminus.

[0053] In certain embodiments of the present invention, the Nucleic Acidligands to tenascin-C described herein are useful for diagnosticpurposes and can be used to image pathological conditions (such as humantumor imaging). In addition to diagnosis, the tenascin-C nucleic acidligands are useful in the prognosis and monitoring of disease conditionsin which tenascin-C is expressed.

[0054] Diagnostic agents need only be able to allow the user to identifythe presence of a given target at a particular locale or concentration.Simply the ability to form binding pairs with the target may besufficient to trigger a positive signal for diagnostic purposes. Thoseskilled in the art would be able to adapt any tenascin-C nucleic acidligand by procedures known in the art to incorporate a marker in orderto track the presence of the nucleic acid ligand. Such a marker could beused in a number of diagnostic procedures, such as detection of primaryand metastatic tumors and athersclerotic lesions. The labeling markerexemplified herein is technetium-99m; however, other markers such asadditional radionuclides, magnetic compounds, fluorophores, biotin, andthe like can be conjugated to the tenascin-C nucleic acid ligand forimaging in an in vivo or ex vivo setting disease conditions in whichtenascin-C is expressed (e.g., cancer, atherosclerosis, and psoriasis).The marker may be covalently bound to a variety of positions on thetenascin-C nucleic acid ligand, such as to an exocyclic amino group onthe base, the 5-position of a pyrimidine nucleotide, the 8-position of apurine nucleotide, the hydroxyl group of the phosphate, or a hydroxylgroup or other group at the 5′ or 3′ terminus of the tenascin-C nucleicacid ligand. In embodiments where the marker is technetium-99m,preferably it is bonded to the 5′ or 3′ hydroxyl of the phosphate groupthereof or to the 5 position of a modified pyrimidine. In the mostpreferred embodiment, the marker is bonded to the 5′ hydroxyl of thephosphate group of the nucleic acid ligand with or without a linker. Inanother embodiment, the marker is conjugated to the nucleic acid ligandby incorporating a pyrimidine containing a primary amine at the 5position, and use of the amine for conjugation to the marker. Attachmentof the marker can be done directly or with the utilization of a linker.In the embodiment where technetium-99m is used as the marker, thepreferred linker is a hexylamine linker as shown in FIG. 2.

[0055] In other embodiments, the tenascin-C nucleic acid ligands areuseful for the delivery of therapeutic compounds (including, but notlimited to, cytotoxic compounds, immune enhancing substances andtherapeutic radionuclides) to tissues or organs expressing tenascin-C.Disease conditions in which tenascin-C may be expressed include, but arenot limited to, cancer, atherosclerosis, and psoriasis. Those skilled inthe art would be able to adapt any tenascin-C nucleic acid ligand byprocedures known in the art to incorporate a therapeutic compound in acomplex. The therapeutic compound may be covalently bound to a varietyof positions on the tenascin-C nucleic acid ligand, such as to anexocyclic amino group on the base, the 5-position of a pyrimidinenucleotide, the 8-position of a purine nucleotide, the hydroxyl group ofthe phosphate, or a hydroxyl group or other group at the 5′ or 3′terminus of the tenascin-C nucleic acid ligand. In the preferredembodiment, the therapeutic agent is bonded to the 5′ amine of thenucleic acid ligand. Attachment of the therapeutic agent can be donedirectly or with the utilization of a linker. In embodiments in whichcancer is the targeted disease, 5-fluorodeoxyuracil or other nucleotideanalogs known to be active against tumors can be incorporated internallyinto existing U's within the tenascin-C nucleic acid ligand or can beadded internally or conjugated to either terminus either directly orthrough a linker. In addition, both pyrimidine analogues2′2′-diFluorocytidine and purine analogues (deoxycoformycin) can beincorporated. In addition, U.S. application Ser. No. 08/993,765, filedDec. 18, 1997, incorporated herein by reference in its entirety,describes, inter alia, nucleotide-based prodrugs comprising nucleic acidligands directed to a tumor, for example tenascin-C, for preciselylocalizing chemoradiosensitizers, and radiosensitizers and radionuclidesand other radiotherapeutic agents to the tumor.

[0056] It is also contemplated that both the marker and therapeuticagent may be associated with the tenascin-C nucleic acid ligand suchthat detection of the disease condition and delivery of the therapeuticagent is accomplished together in one aptamer or as a mixture of two ormore different modified versions of the same aptamer. It is alsocontemplated that either or both the marker and/or the therapeutic agentmay be associated with a non-immunogenic, high molecular weight compoundor lipophilic compound, such as a liposome. Methods for conjugatingnucleic acid ligands with lipophilic compounds or non-immunogeniccompounds in a diagnostic or therapeutic complex are described in U.S.patent application Ser. No. 08/434,465, filed May 4, 1995, entitled“Ligand Nucleic Acid Complexes,” which is incorporated herein in itsentirety.

[0057] The therapeutic or diagnostic compositions described herein maybe administered parenterally by injection (e.g., intravenous,subcutaneous, intradermal, intralesional), although other effectiveadministration forms, such as intraarticular injection, inhalant mists,orally active formulations, transdermal iontophoresis or suppositories,are also envisioned. They may also be applied locally by directinjection, can be released from devices, such as implanted stents orcatheters, or delivered directly to the site by an infusion pump. Onepreferred carrier is physiological saline solution, but it iscontemplated that other pharmaceutically acceptable carriers may also beused. In one embodiment, it is envisioned that the carrier and thetenascin-C nucleic acid ligand complexed with a therapeutic compoundconstitute a physiologically-compatible, slow release formulation. Theprimary solvent in such a carrier may be either aqueous or non-aqueousin nature. In addition, the carrier may contain other pharmacologicallyacceptable excipients for modifying or maintaining the pH, osmolarity,viscosity, clarity, color, sterility, stability, rate of dissolution, orodor of the formulation. Similarly, the carrier may contain still otherpharmacologically-acceptable excipients for modifying or maintaining thestability, rate of dissolution, release, or absorption of the tenascin-Cnucleic acid ligand. Such excipients are those substances usually andcustomarily employed to formulate dosages for parental administration ineither unit dose or multi-dose form.

[0058] Once the therapeutic or diagnostic composition has beenformulated, it may be stored in sterile vials as a solution, suspension,gel, emulsion, solid, or dehydrated or lyophilized powder. Suchformulations may be stored either in ready to use form or requiringreconstitution immediately prior to administration. The manner ofadministering formulations containing tenascin-C nucleic acid ligandsfor systemic delivery may be via subcutaneous, intramuscular,intravenous, intraarterial, intranasal or vaginal or rectal suppository.

[0059] The following examples are provided to explain and illustrate thepresent invention and are not to be taken as limiting of the invention.Example 1 describes the materials and experimental procedures used inExample 2 for the generation of RNA ligands to tenascin-C. Example 2describes the RNA ligands to tenascin-C and the predicted secondarystructure of a selected nucleic acid ligand. Example 3 describes thedetermination of minimal size necessary for high affinity binding of aselected nucleic acid ligand, and substitution of 2′-OH purines with2′-OMe purines. Example 4 describes the biodistribution of Tc-99mlabeled tenascin-C nucleic acid ligands in tumor-bearing mice. Example 5describes the use of a fluorescently labeled tenascin-C nucleic acidligand to localize tenascin-c within tumor tissue.

EXAMPLES Example 1 Use of SELEX to Obtain Nucleic Acid Ligands toTenascin-C and to U251 Glioblastoma Cells

[0060] Materials and Methods

[0061] Tenascin-C was purchased from Chemicon (Temecula, Calif.).Single-stranded DNA-primers and templates were synthesized by OperonTechnologies Inc. (Alameda, Calif.).

[0062] The SELEX-process has been described in detail in the SELEXPatent Applications. In brief, double-stranded transcription templateswere prepared by Klenow fragment extension of 40N7a ssDNA:

[0063] 5′-TCGCGCGAGTCGTCTG[40N]CCGCATCGTCCTCCC3′ (SEQ ID NO:1)

[0064] using the 5N7 primer:

[0065] 5′-TAATACGACTCACTATAGGGAGGACGATGCGG-3′ (SEQ ID NO:2)

[0066] which contains the T7 polymerase promoter (underlined). RNA wasprepared with T7 RNA polymerase as described previously in Fitzwater,T., and Polisky, B. 1996. A SELEX primer. Methods Enzymol. 267, 275-301,incorporated herein by reference in its entirety. All transcriptionreactions were performed in the presence of pyrimidine nucleotides thatwere 2′-fluoro (2′-F) modified on the sugar moiety. This substitutionconfers enhanced resistance to ribonucleases that utilize the2′-hydroxyl moiety for cleavage of the phosphodiester bond.Specifically, each transcription mixture contained 3.3 mM 2′-F UTP and3.3 mM 2′-F CTP along with 1 mM GTP and ATP. The initial randomized RNAlibrary thus produced comprised 3×10¹⁴ molecules. The affinities ofindividual ligands for tenascin-C were determined by standard methodsusing nitrocellulose filter partitioning (Tuerk C, Gold L. Science 1990Aug. 3 ;249(4968):505-10).

[0067] For each round of SELEX, Lumino plates (Labsystems, NeedhamHeights, Mass.) were coated for 2 hours at room temperature with 200 μlDulbecco's PBS containing tenascin-C concentrations as shown in Table 1.After coating, wells were blocked using HBSMC+ buffer [20 mM Hepes, pH7.4, 137 mM NaCl, 1 mM CaCl₂, 1 mM MgCl₂ and 1 g/liter human serumalbumin (Sigma, fraction V) for rounds 1 to 6 while for rounds 7 and 8wells were blocked HBSMC+ buffer containing 1 g/liter casein (I-block;Tropix). Binding and wash buffer consisted of HBSMC+ buffer containing0.05% Tween 20. For each SELEX round, RNA was diluted into 100 μl ofbinding buffer and allowed to incubate for 2 hours at 37° C. in theprotein coated wells that were pre-washed with binding buffer. Afterbinding, six washes of 200 μl each were performed. Following the washstep, the dry well was placed on top of a 95° C. heat block for 5minutes. Standard AMV reverse transcriptase reactions (50 μl) wereperformed at 48° C. directly in the well and the reaction productsutilized for standard PCR and transcription reactions. Two syntheticprimers 5N7 (see above) and 3N7a:

[0068] 5′-TCGCGCGAGTCGTCTG-3′ (SEQ ID NO:3)

[0069] were used for these template amplification and reversetranscription steps.

[0070] For cell SELEX, U251 human glioblastoma cells (Hum. Hered., 1971,21: 238) were grown to confluence in Dulbecco's Modified Eagle's Mediumsupplemented with 10% fetal calf serum (GIBCO BRL, Gaithersburg, Md.) onsix-well tissue culture plates (Becton Dickinson Labware, Lincoln Park,N.J.) and washed three times using Dulbecco's PBS supplemented withCaCl₂ (DPBS, GIBCO BRL) buffer. RNA labeled internally by transcription(Fitzwater, 1996, supra) was incubated with the cells at 37 degrees forone hour. The labeled RNA was then removed, and the cells were washedsix times for ten minutes each at 37 degrees with DPBS. DPBS containing5 mM EDTA was then added and incubated with the cells for 30 minutes toelute bound RNAs that remained after the washing steps. This RNA wasquantitated by a standard liquid scintillation counting protocol andamplified using RT-PCR.

[0071] Binding assays for the U251 cells. Internally labeled RNA wasincubated at increasing concentrations with confluent U251 cells insix-well tissue culture plates (Becton Dickinson Labware, Lincoln Park,N.J.) at 37 degrees for 60 min. Unbound RNA was washed away using three10 minute washes with DPBS+ CaCl₂ at 37 degrees, and bound RNA wascollected by disrupting the cells using Trizol (Gibco BRL, Gaithersburg,Md.). Bound RNA was quantitated by liquid scintillation counting.

[0072] Cloning and Sequencing. Amplified affinity enrichedoligonucleotide pools were purified on an 8% polyacrylamide gel, reversetranscribed into ssDNA and the DNA amplified by the polymerase chainreaction (PCR) using primers containing BamH1 and HindIII restrictionendonuclease sites. PCR fragments were cloned, plasmids prepared andsequence analyses performed according to standard techniques (Sambrooket al. (1989) Molecular Cloning: A Laboratory Manual, 2^(nd) Ed. 3vols., Cold Spring Harbor Laboratory Press, Cold Spring Harbor).

Example 2 RNA Ligands to Tenascin-C

[0073] Nucleic Acid Ligands to U251 cells were obtained by the SELEXprocess and are described in U.S. patent application Ser. No.08/434,425, entitled “Systematic Evolution of Ligands by ExponentialEnrichment: Tissue SELEX,” filed May 3, 1995, now U.S. Pat. No.5,789,157. Subsequently it was determined that the ligands that wereobtained were tenascin-C nucleic acid ligands.

[0074] To obtain oligonucleotide ligands against human tenascin-C, eightrounds of SELEX were performed using the randomized nucleotide libraryas described above in Materials and Methods. RNA and protein input intoeach round is shown in Table 1. After 8 rounds of SELEX, the affinity ofthe oligonucleotide pool for tenascin-C was 10 nM, and this affinity didnot increase with additional SELEX rounds.

[0075] To obtain ligands to U251 glioblastoma cells, nine rounds ofSELEX were performed using the randomized nucleotide library. After ninerounds of binding to U251 cells and EDTA elution, rounds 3, 5 and 9 weretested for their ability to bind to U251 cells. FIG. 1 shows that as thenumber of SELEX rounds increases, the amount of bound RNA also increasesat a particular concentration. Because of the complexity of the targettissue, it was not possible to estimate the affinity of theoligonucleotide pools for the unknown target molecules(s) on thesecells.

[0076] The E9 pool (nine rounds of binding and EDTA elution from U251cells) was then used as a starting point for a SELEX against purifiedtenascin-C. Two rounds of SELEX using purified tenascin-C were performedas described above. Input protein and RNA concentrations for two roundsof SELEX (E9P1 and E9P2) are described in Table 2.

[0077] In summary, three different SELEX experiments were performed: anexperiment using purified tenascin-C as the target, an experiment usingU251 glioblastoma cells as the target, and an experiment in which theSELEX pool from the U251 glioblastoma cells was used to initiate a SELEXexperiment using purified tenascin-C as the target.

[0078] All three SELEX experiments were analyzed by cloning andsequencing ligands from round 8 of the purified tenascin-C SELEX (“TN”sequences), from round 9 of the U251 cell SELEX (“E9” sequences), andfrom round 2 of the U251/tenascin-C hybrid SELEX (“E9P2” sequences). Thesequences of 34 unique clones are shown in Table 3, and are divided intotwo major groups: tenascin-C ligands (“TN” and “E9P2” sequences) andU251 cell ligands (“E9” ligands). Among the tenascin-C ligands, themajority of the clones (65 total) represent one of two distinct sequenceclasses designated Family I and Family II (FIG. 1). Examination of thevariable region of the 12 clones in Family I revealed 7 unique sequencesthat are related through the consensus sequence GACNYUUCCNGCYAC (SEQ IDNO: 12). Examination of the variable region of the 18 clones in FamilyII revealed sequences that share a consensus sequence CGUCGCC (Table3;). The E9 sequences could be grouped into a related set by virtue ofconserved GAY and CAU sequences within the variable regions. Theremaining sequences did not appear related to other sequences and wereclassified as orphans. Three sequences predominate, with E9P2-1, E9P2-2,and TN9 represented 14, 16, and 10 times respectively. In the “Orphan”category, one sequence, TN18, was represented twice. Overall, these datarepresent a highly enriched sequence pool.

[0079] Most individuals displayed low nanomolar dissociation constants,with the three most prevalent sequences, TN9 and E9P2-1 and -2, havingthe highest affinities at 5 nM, 2 nM, and 8 nM. (Table 3). These resultsindicate that the U251 cell SELEX is a repository for aptamers againsttenascin-C, and that only two rounds of SELEX were required to isolatethe tenascin-specific ligands from the cell SELEX pool. Oligonucleotideligands against other proteins can be similarly isolated from the E9pool using purified protein targets.

Example 3 Determination of Minimal Size of TN9, and Substitution of2′-OH Purines with 2′-OMe Purines: Synthesis of Aptamer TTA1

[0080] Oligonucleotide synthesis procedures were standard for thoseskilled in the art (Green L S, Jellinek D, Bell C, Beebe L A, Feistner BD, Gill S C, Jucker F M, Janjic N. Chem Biol 1995 October;2(10):683-95). 2′-fluoro pyrimidine phosphoramidite monomers wereobtained from JBL Scientific (San Luis Obispo, Calif.); 2′-OMe purine,2′-OH purine, hexyl amine, and (CH₂CH₂O)₆ monomers, along with the dTpolystyrene solid support, were obtained from Glen Research (Sterling,Va.). Aptamer affinities were determined using nitrocellulose filterpartitioning (Green et al., supra).

[0081] TN9 was chosen for further analysis based on its high affinityfor tenascin-C. We first searched for a minimal sequence necessary forhigh affinity binding. Using standard techniques (Green et al, supra),it was discovered that nucleotides 3′ of nucleotide 55 were required forbinding to tenascin-C, while no nucleotides could be removed from the 5′end without loss of affinity. To further decrease the TN9's length from55 nucleotides and retain high affinity binding, we then attempted todefine internal deletions of TN9. The first 55 nucleotides of TN9, alongwith the first 55 nucleotides of related family II ligands TN7, TN21,and TN41, were input into a computer algorithm to determine possible RNAsecondary structure foldings (mfold 3.0, accessed athttp://www.ibc.wustl.edu/˜zuker/. M. Zuker, D. H. Mathews & D. H.Turner. Algorithms and Thermodynamics for RNA Secondary StructurePrediction: A Practical Guide. In: RNA Biochemistry and Biotechnology,J. Barciszewski & B. F. C. Clark, eds., NATO ASI Series, Kluwer AcademicPublishers, (1999)). Among many potential RNA foldings predicted by thealgorithm, a structure common to each oligonucleotide was found. Thisstructure, represented by oligonucleotide TTA1 in FIG. 2, contains threestems that meet at a single junction, a so-called 3-stem junction. Thisfolding places the most highly conserved nucleotides of family IIoligonucleotides at the junction area. In comparing TN9, TN7, TN21, andTN41, the second stem was of variable length and sequence, suggestingthat extension of the second stem is not required for binding totenascin-C. Testing this hypothesis on TN9, we found that nucleotides10-26 could be replaced with an ethylene glycol linker, (CH₂CH₂O)₆. Thelinker serves as a substitute loop and decreases the size of theaptamer. Additionally, four-nucleotide loops (CACU or GAGA) that replacenucleotides 10-26 produce sequences with high affinity for tenascin-C.It would be well within one skilled in the art to determine othernucleotide loops or other spacers that could replace nucleotides 10-26to produce sequences with high affinity for tenascin-C.

[0082] To increase protection against nuclease activity, purinepositions that could be substituted with the corresponding 2′-OMepurines were located. The oligonucleotide was arbitrarily divided intofive sectors and all purines within each sector were substituted by thecorresponding 2′-OMe purine nucleotide, a total of five oligonucleotides(Table 4, Phase I syntheses). The affinity of each oligonucleotide fortenascin-C was determined, and it was found that all purines withinsectors 1, 3 and 5 could be substituted without appreciable loss inaffinity. Within sectors 2 and 4, individual purines were thensubstituted with 2′-OMe purines and the effect of affinity was measured(Table 4, Phase III syntheses). From these experiments, it was deducedthat substitution of nucleotides G9, G28, G31, and G34, with 2′-OMe Gcauses loss in affinity for tenascin-C. Therefore these nucleotidesremain as 2′-OH purines in the aptamer TTA1.

[0083] The aptamer TTA1 (Table 4) was then synthesized with the(CH₂CH₂O)₆ (Spacer 18) linker, a 3′-3′ dT cap for exonucleaseprotection, a 5′ hexyl amine (Table 4), and all purines as 2′-OMe exceptthe 5 Gs indicated in Table 4. A non-binding control aptamer, TTA1.NB,was generated by deleting 5 nucleotides at the 3′ end to produceTTA1.NB. TTA1 binds to tenascin-C with an equilibrium dissociationconstant (K_(d)) of 5 nM, while TTA1.NB has a K_(d) of >5 μM fortenascin-C.

[0084] Nucleotides 10-26 can be replaced by a non-nucleotide ethyleneglycol linker. It is therefore likely that TTA1 can be synthesized intwo separate pieces, where a break is introduced at the position of theethylene glycol linker and new 5′ and 3′ ends are introduced. Subsequentto synthesis, the two molecules will incubated together to allow hybridformation. This method allows introduction of additional amine groups aswell as nucleotides at the new 5′ and 3 ends. The new functionalitiescould be used for bioconjugation. In addition, two-piece synthesisresults in increased chemical synthetic yield due to shortening thelength of the molecules.

Example 4 Biodistribution of Tc-99m Labeled Aptamers in Tumor-BearingMice

[0085] Aptamer biodistribution was tested by conjugating a Tc-99mchelator (Hi₁₅; Hilger, C. S., Willis, M. C., Wolters, M. Pieken, W. A.1998. Tet, Lett 39, 9403-9406) to the 5′ end of the oligonucleotide asshown in FIG. 2, and radiolabeling the aptamer with Tc-99m. TTA1 andTTA1.NB were conjugated to Hi₁₅ at 50 mg/ml aptamer in 30%dimethylformamide with 5 molar equivalents of Hi₁₅-N-hydoxysuccinimide,buffered in 100 mM Na Borate pH 9.3, for 30 minutes at room temperature.Reversed phase HPLC purification yielded Hi₁₅-TTA1 and Hi₁₅-TTA1.NB. Theoligonucleotides were then labeled with Tc-99m in the following manner:to 1 nmole Hi15-aptamer was added 200 μL of 100 mM NaPO4 buffer, pH 8.5,23 mg/mL NaTartrate, and 50 μL Tc-99m pertechnetate (5.0 mCi) elutedfrom a Mo-99 column (Syncor, Denver) within 12 hours of use. Thelabeling reaction was initiated by the addition of 10 μL 5 mg/mL SnCl₂.The reaction mixture was incubated for 15 minutes at 90° C. The reactionwas separated from unreacted Tc-99m by spin dialysis through a 30,000 MWcut-off membrane (Centrex, Schleicher & Scheull) with two 300 μL washes.This labeling protocol results in 30-50% of the added 99mTc beingincorporated with a specific activity of 2-3 mCi/nmole RNA. The Tc-99mis bound through the 5′G as shown in FIG. 5.

[0086] For biodistribution experiments, U251 xenograft tumors wereprepared as follows: U251 cells were cultured in Dulbeccos' ModifiedEagle's Medium supplemented with 10% v/v fetal calf serum (Gibco BRL,Gaithersburg, Md.). Athymic mice (Harlan Sprague Dawley, Indianapolis,Ind.) were injected subcutaneously with 1×10⁶ U251 cells. When thetumors reached a size of 200-300 mg (1-2 weeks), Tc-99m labeled aptamerwas injected intravenously at 3.25 mg/kg. At indicated times, animalswere anesthetized using isoflurane (Fort Dodge Animal Health, FortDodge, Iowa), blood was collected by cardiac puncture, and the animalwas sacrificed and tissues were harvested. Tc-99m levels were countedusing a gamma counter (Wallac Oy, Turku, Finland). Aptamer uptake intotissues was measured as the % of injected dose per gram of tissue (%ID/g).

[0087] Images of mice were obtained using a gamma camera. Mice wereplaced onto the camera (Siemens, LEM+) under anesthesia (isoflurane).Data were collected (30 sec to 10 minutes) and analyzed using NuclearMAC software version 3.22.2 (Scientific Imaging, CA) on a Power MAC G3(Apple Computer, CA).

[0088] Biodistribution experiments, Table 5, indicated rapid andspecific uptake of the aptamer into tumor tissue; the non-bindingaptamer does not remain in the tumor. Blood levels of Tc-99m alsocleared rapidly. After three hours, Tc-99m levels brought into the tumorusing Hi₁₅-TTA1 had a very long half life (>18 hrs). This indicates thatonce the aptamer penetrates the tumor, the radiolabel carried with itremains in the tumor for long periods of time. Such data indicate thatcytotoxic agents, including radionuclides and non-radioactive agents,conjugated to the aptamer will also remain in the tumor with long halflives.

[0089] Tc-99m radioactivity also appears in other tissues, notably thesmall and large intestines. The hepatobiliary clearance pattern seenhere can be readily altered by those skilled in the art, for example byaltering the hydrophilicity of the Tc-99m chelator, changing thechelator, or changing the radiometal/chelator pair altogether.

[0090] Whole animal images were obtained using Tc-99m labeled Hi₁₅-TTA1and at 3 hours post-injection. Images obtained from mice injected withHi₁₅-TTA1, but not from mice injected with Hi₁₅-TTA1.NB, clearly showthe tumor (FIG. 3). Additional radioactivity is evident ingastrointestinal tract, as predicted by the biodistribution experiments.

Example 5 Use of Fluorescently Labeled TTA1 to Localize Tenascin-CWithin Tumor Tissue

[0091] Materials and Methods.

[0092] TTA1 and TTA1.NB were synthesized as described above. SuccinimdylRhodamine-Red-X (Molecular Probes, Eugene, Oreg.) was conjugated to the5′ amine of the aptamers as described above for H₁₅-NHS conjugation. TheRhodamine-Red-X-conjugated aptamers, TTA1-Red and TTA1.NB-Red, werepurified by reversed phase HPLC. U251 cell culture and tumor growth innude mice were as described above. Five nmol of TTA1-Red or TTA1.NB-Redwere injected intravenously into nude mice and at the desired time theanimal was placed under anesthesia, perfused with 0.9% NaCl, andsacrificed. The tumor was excised and placed in formalin. After 24 hr informalin, 10 μM sections were cut and Rhodamine-Red-X was detected usinga fluorescence microscope (Eclipse E800, Nikon, Japan).

[0093] Results: TTA1-Red has identical affinity for tenascin-C as theunconjugated parent aptamer, TTA1, at 5 nM. We compared tumorfluorescence levels of TTA1-Red and TTA1.NB-Red 10 min post-injection.The binding aptamer, TTA1-Red, strongly stains the tumor but notadjacent tissue (FIG. 4). In contrast, only tissue auto-fluorescence isdetected with TTA1.NB-Red. These results demonstrate the utility of theaptamer in fluorescent detection of tenascin-C in vivo, and the aptamermay be similarly used for staining tissues sections ex vivo. TABLE 1Tenascin-C SELEX RNA and protein input. Tenascin-C RNA Round (pMol/well)(pMol/well) 1 12 200 2 12 200 3 12 200 4 12 200 5 2 33 6 2 33 7 2 33 80.2 3.3

[0094] TABLE 2 Cell SELEX/tenascin-C SELEX RNA and protein inputTenascin-C RNA Round (pMol/well) (pMol/well) E9P1 2 33 E9P2 2 33

[0095] TABLE 3 Tenascin-C Sequences: purified protein SELEX (tenascinsequences) and U251 cell SELEX + purified protein SELEX (E9P2 sequences)SEQ ID NO: Family I TN11 4 ggGAggAcGauGcgg CAAUcAAAACUcACGUUA UUCCCUCAUUCUAUUAGCUUCCC cagacgacucgcccga 10 nM TN45 5 qggaggacgaugcggCAAUCUcCGAAAAAGACUCUUCCU GCAUCCUCUcACCCCC cagacgacucgcccga 30 nM TN4 6gggaggacgaugcgg CAACCUc   GAAAGACUUUUCCC GCAUCACUGUGUACUCCCCcagacgacucgcccga 40 nM TN22 7 gggaggacgaugcgg CAACCUc   GAUAGACUUUUCCCGCAUCACUGUGUACUCCCC cagacgacucgcccga 40 nM TN32(2) 8 gggAggAcgauCcggcAaCCUcAA UCUuGaCAUUUCCC GcACCUAAAUUUG  CCCC cagacgacucgcccga 15 nM TN149 gggaggacgaugcgg CAAACGAUC ACU UACCUUUCCU GCAUCUGCUAGC CUCCCCcagacgacucgcccga 20 nM TN44(3) 10 gggaggacgaugcgg  ACGCCAGCCAUUGACCCUCGCUUCCACUAUUCCAUCCCCC cagacgacucgcccga 10 nMTN29(2) 11 gggaggacgaugcgg CCAACCUCAUUUUGACACUUCGCCGCACCUAAUUGCCCCcagacgacucgcccga 25 nM consensus: 12                              GACNYUUCCN GCAYC Family II E922-4(5) 13gggaggacgaugcgg AACCCAUA ACGCGA ACCGACCAACAUGCCUCCCGUGCCCCcagacgacucgcccga E9P2-1(14) 14 gggAggacgaugcgg UGCCCAUAGAAGCGU GCCGCUAAUGCUAACGCCCUCCCC cagacgacucgcccga 2 nM E9P2-2(16) 15gggaggacgaugcgg UGCCCACU AUGCGU GCCGAAAAACAUUUCCCCCUCUACCCcagacgacucgcccga 8 nM TN7 (3) 16 gggaggacgaugcggAACACUUUCCCAUGCGUCGCC AUACC GGAUAUAUUGCUCC cagacgacucgcccga 20 nMTN21(4) 17 gggaggacgaugcgg   ACUGGACCAAACCGUCGCCGAUACCCGGAUACUUUGCUCCcagacgacucgcccga 10 nM TN9(10) 18 gggaggacgaugcgg   AACAAUGCACUCGUCGCCGUAAU GGAUGUUUUGCUCCCUG cagacgacucgcccga 5 nM TN4119 gggaggacgaugcgg UUAAGUCUCGGUUGAAU GCCCAUCCC AGAUCCCCCUGACCcagacgacucgcccga 20 nM consensus:                              GCGUCGCCG Orphans E992-17 20gggaggacgaugcgg AUGGCAAGUCGAACCAUCCCCCACGCUUCUCCUGUUCCCCcagacgacucgcccga E992-48 21 gggaggacgaugcggGAAGUUUUcUCUGCCUUGGUUUCGAUUGGCGCCUccCCCC cagacgacucgcccga1 E9P2-14 22gggaggacgaugcgg UCGAGCGgUCGACCGUCAACAAGAAUAAAGCGUGUCCCUGcagacgacucgcccga E9P2-17 23 gggaggacgaugcggAUGGCAAGUCGAACCAUCCCCCACGCUUCUCCUGUUCCCC cagacgacucgcccga E9P2-22 24gggaggacgaugcgg ACUAGACcgCGAGUCCAUUCAACUUGCCCAAAAaAAAACcUCCCCcagacgacucgcccga E9P2-40 25 gggaggacgaugcggGAGAUCAACAUUCCUCUAGUUUGGUUCCAACCUACACCCC cagacgacucgcccga E9P2-41 26gggaggacgaugcgg ACGAGCGUCUCAUGAUCACACUAUUUCGUCUCAGUGUGCAcagacgacucgcccga TNT8 27 gggaggacgaugcggUCGACCUCGAAUGACUCUCCACCUAUCUAACAUCCCCCCC cagacgacucgcccga 145 nM TN20 28gggaggacgaugcgg UCGACCUCGAAUGACUCUCCACCUAUCUAACAGCCUUCCCcagacgacucgcccga TN5T 29 gggaggacgaugcggAGAACUCAUCCUAACCGCUCUAACAAAUCUUGUCCGACCG cagacgacucgcccga TN8 30gggaggacgaugcgg AUAAUUcGACACCAACCAGGUCCCGGAAAUCAUCCCUCUGcagacgacucgcccga >10 uM TN27 31 gggaggacgaugcgg AAACCAACCGUUGACCACCUUUUCGUUUCCGGAAAGUCCC cagacgacucgcccga 110 nM TN39 32 gggaggacgaugcggAAGCCAACCCUCUAGUCAGCCUUUCGUUUCCCACGCCACC cagacgacucgcccga TN24 33gggaggacgaugcGg gACCAACUAAACUGUUCGAAAGCUGGaACAUGUCCUGACGCcagacgacucgcccga 10 nM TN5 34 gggaggacgaugcggACCAACUAAACUGUUCGAAAGCUGGAACACGUCCUGACGC cagacgacucgcccga TN3G 35gggaggacgaugcgg ACCAACUAAACUGUUCGAAAGCUAGAACACGUCCAGACGCcagacgacucgcccga TN36 36 gggaggacgaugcggACCAACUAAACUGUUCGAAAGCUGGAACACGUUCUGACGC cagacgacucgcccga TN10 37gggaggacgaugcgg ACCAACUAAACUGUUCGAAAGCUGGAAUACGUCCUGACGCcagacgacucgcccga TN1 38 gggaggacgaugcgg AAGUUUAGuGCUCCAGUUCCGACACUCCUcUACUCAGCCC cagacgacucgcccga >10 uM TN109 39qggaggacgaugcgG AgCCAGAGCCUcUcUcAGUUcUaCAGAACUuACCcACUGGcagacgacucgcccga TN110 40 gggaggacgaugcggACCUAACUCAAUCAGGAACCAAACCUAGCACUCUCAUGGC cagacgacucgcccga U251 SELEXAptamers, EDTA Elution (E9) E9-8(3) 41 gggaggacgaugcgg   GAGAUCAACAUUCCUCUAGUUUGGUUCCCAACCUACACCCC cagacgacucgcccga E9-15 42gggaggacgaugcgg AUCUCGAUCCUUCAGCACUUCAUUUCAUUCCUUUcUGCCCcagacgacucgcccga E9-6 43 gggaggacgaugcgg    ACGAUCCUUUCCUUACAUUUCAUCAUUUCUCUUGUGCCC cagacgacucgcccga E9-5(2) 44 gggaggacgaugcggUGACGACAACUCGACUG CAUAUCUCACAACUCCUGUGCCC cagacgacucgcccga E9-3(6) 45gggaggacgaugcgg ACUAGACCGCGAGUC   CAUUCAACUUGCCCAAAAACCUCCCCcagacgacucgcccga E9-9 46 gggaggacgaugcgg      GCGCAUCGACCAACAUCCGAUUCGGAUUCCUCCACUCCCC cagacgacugcccga

[0096] TABLE 4 2′-OMe Substitutions, Internal Deletions, TTA1, andTTA1.NB Sequence SEQ ID NO: Kd Phase I. 2′-OMe.Affinity. TN9.3 47gggaggacgaugcggAACAAUGCACUCGUCGCCGUAAUGGAUGUUUUGCU5 >10 uM TN9.4 48GGGAGGACGAUGCGGAACAAUGCACUCGUCGCCGUAAUGGAUGUUUUGCUCCCUG5 2 nM TN9.4M1 4966676GACGAUGCGGAACAAUGCACUCGUCGCCGUAAUGGAUGUUUUGCUCCCU65 6 nM TN9.4M2 50GGGAG67C67U6C6GAACAAUGCACUCGUCGCCGUAAUGGAUGUUUUGCUCCUG5 20 nM TN9.4M3 51GGGAGGACGAUGCG677C77U6C7CUCGUCGCCGUAAUGGAUGUUUUGCUCCCUG5 7 nM TN9.4M4 52GGGAGGACGAUGCGGAACAAUGCACUC6UC6CC6UAAUGGAUGUUUUGCUCCCUG5 nb TN9.4M5 53GGGAGGACGAUGCGGAACAAUGCACUCGUCGCCGU77U667U6UUUU6CUCCCUG5 4 nM TN9.4Me 5416667667C67U6C6677C77U6C7CUC6UC6CC6U77U667U6UUUU6CUCCCU65 10 nM PhaseIII. 2′-OMe.Affinity. TN9.4M1235 5516667667C67U6C6677C77U6C7CUCGCUCGCCGU77U667U6UUUU6CUCCCU65 16.5 nMTN9.4M135G6 56 1666766ACGAUGCG677C77U6C7CUCGUCGCCGU77U667U6UUUU6CUCCCU652.2 nM TN9.4M135A7 57166676G7CGAUGCG677C77U6C7CUCGUCGCCGU77U667U6UUUU6CUCCCU65 1.7 nMTN9.4M135G9 58 166676GAC6AUGCG677C77U6C7CUCGUCGCCGU77U667U6UUUU6CUCCCU657.7 nM TN9.4M135A10 59166676GACG7UGCG677C77U6C7CUCGUCGCCGU77U667U6UUUU6CUCCCU65 1.3 nMTN9.4M135G12c14 60166676GACGAU6C6677C77U6C7CUCGUCGCCGU77U667U6UUUU6CUCCCU65 2.5 nMTN9.4M135G28 61166676GACGAUGCG677C77U6C7CUC6UCGCCGU77U667U6UUUU6CUCCCU65 37 nMTN9.4M135G31 62166676GACGAUGCG677C77U6C7CUCGUCGCCGU77U667U6UUUU6CUCCCU65 55 nMTN9.4M135G34 63166676GACGAUGCG677C77U6C7CUCGUCGCCGU77U667U6UUUU6CUCCCU65 7 nM TTA1: 645′-1G667667CG-(CH₂CH₂O)₆-CGUCGCCGU77U667U6UUUU6CUCCCU65 5 nM TTA1.NB: 655′-1G667667CG-(CH₂CH₂O)₆-CGUCGCCGU77U667U6UUUU6CU5 >5 uM

[0097] TABLE 5 Biodistribution of Tc-99m-TTA1 and -TTA1.NB min TTA1TTA1.NB tumor 2 4.470 ± 0.410 4.510 ± 0.300 10 5.940 ± 0.590 3.020 ±0.210 60 2.689 ± 0.310 0.147 ± 0.018 180 1.883 ± 0.100 0.043 ± 0.004 5701.199 ± 0.066 0.018 ± 0.001 1020 1.150 ± 0.060 N/A blood 2 18.247 ±1.138  15.013 ± 0.506  10 2.265 ± 0.245 2.047 ± 0.195 60 0.112 ± 0.0030.102 ± 0.019 180 0.032 ± 0.001 0.034 ± 0.003 570 0.013 ± 0.001 0.011 ±0.001 1020 0.006 ± 0.001 N/A lung 2 8.970 ± 1.210 8.130 ± 0.960 10 2.130± 0.080 1.940 ± 0.230 60 0.157 ± 0.011 0.120 ± 0.005 180 0.048 ± 0.0060.041 ± 0.003 570 0.028 ± 0.006 0.017 ± 0.002 1020 0.007 ± 0.001 N/Aliver 2 9.120 ± 0.530 7.900 ± 0.350 10 12.460 ± 1.250  9.100 ± 0.830 601.234 ± 0.091 0.423 ± 0.095 180 0.401 ± 0.084 0.211 ± 0.059 570 0.104 ±0.017 0.058 ± 0.003 1020 0.075 ± 0.003 N/A spleen 2 5.100 ± 0.410 4.860± 0.130 10 2.460 ± 0.210 1.220 ± 0.120 60 0.643 ± 0.076 0.110 ± 0.015180 0.198 ± 0.026 0.038 ± 0.005 570 0.062 ± 0.004 0.020 ± 0.001 10200.030 ± 0.003 N/A kidney 2 44.430 ± 4.280  54.470 ± 1.210  10 18.810 ±0.940  14.320 ± 2.080  60 1.514 ± 0.040 0.637 ± 0.111 180 0.286 ± 0.0280.221 ± 0.021 570 0.140 ± 0.006 0.100 ± 0.013 1020 0.081 ± 0.005 N/A sm.int. 2 3.690 ± 0.250 3.120 ± 0.100 10 7.010 ± 0.070 6.440 ± 0.250 6015.716 ± 2.036  14.649 ± 0.532  180 1.479 ± 0.710 1.243 ± 0.405 5700.219 ± 0.147 0.159 ± 0.067 1020 0.280 ± 0.243 N/A lg. int. 2 2.340 ±0.240 2.280 ± 0.180 10 0.890 ± 0.040 0.770 ± 0.070 60 10.799 ± 5.381 21.655 ± 11.676 180 26.182 ± 7.839  18.023 ± 3.485  570 1.263 ± 0.7060.716 ± 0.179 1020 0.298 ± 0.167 N/A muscle 2 1.270 ± 0.130 1.490 ±0.050 10 0.870 ± 0.090 1.840 ± 1.000 60 0.064 ± 0.003 0.050 ± 0.004 1800.016 ± 0.002 0.011 ± 0.001 570 0.011 ± 0.002 0.007 ± 0.001 1020  0.003± 0.0003

[0098]

1 65 1 71 DNA Artificial Sequence Description of Artificial SequenceSynthetic Sequence 1 tcgcgcgagt cgtctgnnnn nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnccgc 60 atcgtcctcc c 71 2 32 DNA Artificial SequenceDescription of Artificial Sequence Synthetic Sequence 2 taatacgactcactataggg aggacgatgc gg 32 3 16 DNA Artificial Sequence Description ofArtificial Sequence Synthetic Sequence 3 tcgcgcgagt cgtctg 16 4 71 RNAArtificial Sequence Description of Artificial Sequence SyntheticSequence 4 gggaggacga ugcggcaauc aaaacucacg uuauucccuc aucuauuagcuuccccagac 60 gacucgcccg a 71 5 71 RNA Artificial Sequence Descriptionof Artificial Sequence Synthetic Sequence 5 gggaggacga ugcggcaaucuccgaaaaag acucuuccug cauccucuca ccccccagac 60 gacucgcccg a 71 6 71 RNAArtificial Sequence Description of Artificial Sequence SyntheticSequence 6 gggaggacga ugcggcaacc ucgaaagacu uuucccgcau cacuguguacucccccagac 60 gacucgcccg a 71 7 71 RNA Artificial Sequence Descriptionof Artificial Sequence Synthetic Sequence 7 gggaggacga ugcggcaaccucgauagacu uuucccgcau cacuguguac ucccccagac 60 gacucgcccg a 71 8 71 RNAArtificial Sequence Description of Artificial Sequence SyntheticSequence 8 gggaggacga ugcggcaacc ucaaucuuga cauuucccgc accuaaauuugcccccagac 60 gacucgcccg a 71 9 71 RNA Artificial Sequence Descriptionof Artificial Sequence Synthetic Sequence 9 gggaggacga ugcggcaaacgaucacuuac cuuuccugca ucugcuagcc ucccccagac 60 gacucgcccg a 71 10 71 RNAArtificial Sequence Description of Artificial Sequence SyntheticSequence 10 gggaggacga ugcggacgcc agccauugac ccucgcuucc acuauuccauccccccagac 60 gacucgcccg a 71 11 70 RNA Artificial Sequence Descriptionof Artificial Sequence Synthetic Sequence 11 gggaggacga ugcggccaaccucauuuuga cacuucgccg caccuaauug cccccagacg 60 acucgcccga 70 12 15 RNAArtificial Sequence Description of Artificial Sequence SyntheticSequence 12 gacnyuuccn gcayc 15 13 71 RNA Artificial SequenceDescription of Artificial Sequence Synthetic Sequence 13 gggaggacgaugcggaaccc auaacgcgaa ccgaccaaca ugccucccgu gcccccagac 60 gacucgcccg a71 14 70 RNA Artificial Sequence Description of Artificial SequenceSynthetic Sequence 14 gggaggacga ugcggugccc auagaagcgu gccgcuaaugcuaacgcccu cccccagacg 60 acucgcccga 70 15 71 RNA Artificial SequenceDescription of Artificial Sequence Synthetic Sequence 15 gggaggacgaugcggugccc acuaugcgug ccgaaaaaca uuucccccuc uaccccagac 60 gacucgcccg a71 16 71 RNA Artificial Sequence Description of Artificial SequenceSynthetic Sequence 16 gggaggacga ugcggaacac uuucccaugc gucgccauaccggauauauu gcucccagac 60 gacucgcccg a 71 17 71 RNA Artificial SequenceDescription of Artificial Sequence Synthetic Sequence 17 gggaggacgaugcggacugg accaaaccgu cgccgauacc cggauacuuu gcucccagac 60 gacucgcccg a71 18 71 RNA Artificial Sequence Description of Artificial SequenceSynthetic Sequence 18 gggaggacga ugcggaacaa ugcacucguc gccguaauggauguuuugcu cccugcagac 60 gacucgcccg a 71 19 71 RNA Artificial SequenceDescription of Artificial Sequence Synthetic Sequence 19 gggaggacgaugcgguuaag ucucgguuga augcccaucc cagauccccc ugacccagac 60 gacucgcccg a71 20 71 RNA Artificial Sequence Description of Artificial SequenceSynthetic Sequence 20 gggaggacga ugcggauggc aagucgaacc aucccccacgcuucuccugu ucccccagac 60 gacucgcccg a 71 21 71 RNA Artificial SequenceDescription of Artificial Sequence Synthetic Sequence 21 gggaggacgaugcgggaagu uuucucugcc uugguuucga uuggcgccuc ccccccagac 60 gacucgcccg a71 22 71 RNA Artificial Sequence Description of Artificial SequenceSynthetic Sequence 22 gggaggacga ugcggucgag cggucgaccg ucaacaagaauaaagcgugu cccugcagac 60 gacucgcccg a 71 23 71 RNA Artificial SequenceDescription of Artificial Sequence Synthetic Sequence 23 gggaggacgaugcggauggc aagucgaacc aucccccacg cuucuccugu ucccccagac 60 gacucgcccg a71 24 76 RNA Artificial Sequence Description of Artificial SequenceSynthetic Sequence 24 gggaggacga ugcggacuag accgcgaguc cauucaacuugcccaaaaaa aaaccucccc 60 cagacgacuc gcccga 76 25 71 RNA ArtificialSequence Description of Artificial Sequence Synthetic Sequence 25gggaggacga ugcgggagau caacauuccu cuaguuuggu uccaaccuac acccccagac 60gacucgcccg a 71 26 71 RNA Artificial Sequence Description of ArtificialSequence Synthetic Sequence 26 gggaggacga ugcggacgag cgucucaugaucacacuauu ucgucucagu gugcacagac 60 gacucgcccg a 71 27 71 RNA ArtificialSequence Description of Artificial Sequence Synthetic Sequence 27gggaggacga ugcggucgac cucgaaugac ucuccaccua ucuaacaucc ccccccagac 60gacucgcccg a 71 28 71 RNA Artificial Sequence Description of ArtificialSequence Synthetic Sequence 28 gggaggacga ugcggucgac cucgaaugacucuccaccua ucuaacagcc uuccccagac 60 gacucgcccg a 71 29 71 RNA ArtificialSequence Description of Artificial Sequence Synthetic Sequence 29gggaggacga ugcggagaac ucauccuaac cgcucuaaca aaucuugucc gaccgcagac 60gacucgcccg a 71 30 71 RNA Artificial Sequence Description of ArtificialSequence Synthetic Sequence 30 gggaggacga ugcggauaau ucgacaccaaccaggucccg gaaaucaucc cucugcagac 60 gacucgcccg a 71 31 71 RNA ArtificialSequence Description of Artificial Sequence Synthetic Sequence 31gggaggacga ugcggaaacc aaccguugac caccuuuucg uuuccggaaa guccccagac 60gacucgcccg a 71 32 71 RNA Artificial Sequence Description of ArtificialSequence Synthetic Sequence 32 gggaggacga ugcggaagcc aacccucuagucagccuuuc guuucccacg ccacccagac 60 gacucgcccg a 71 33 72 RNA ArtificialSequence Description of Artificial Sequence Synthetic Sequence 33gggaggacga ugcgggacca acuaaacugu ucgaaagcug gaacaugucc ugacgccaga 60cgacucgccc ga 72 34 71 RNA Artificial Sequence Description of ArtificialSequence Synthetic Sequence 34 gggaggacga ugcggaccaa cuaaacuguucgaaagcugg aacacguccu gacgccagac 60 gacucgcccg a 71 35 71 RNA ArtificialSequence Description of Artificial Sequence Synthetic Sequence 35gggaggacga ugcggaccaa cuaaacuguu cgaaagcuag aacacgucca gacgccagac 60gacucgcccg a 71 36 71 RNA Artificial Sequence Description of ArtificialSequence Synthetic Sequence 36 gggaggacga ugcggaccaa cuaaacuguucgaaagcugg aacacguucu gacgccagac 60 gacucgcccg a 71 37 71 RNA ArtificialSequence Description of Artificial Sequence Synthetic Sequence 37gggaggacga ugcggaccaa cuaaacuguu cgaaagcugg aauacguccu gacgccagac 60gacucgcccg a 71 38 71 RNA Artificial Sequence Description of ArtificialSequence Synthetic Sequence 38 gggaggacga ugcggaaguu uagugcuccaguuccgacac uccucuacuc agccccagac 60 gacucgcccg a 71 39 71 RNA ArtificialSequence Description of Artificial Sequence Synthetic Sequence 39gggaggacga ugcggagcca gagccucucu caguucuaca gaacuuaccc acuggcagac 60gacucgcccg a 71 40 71 RNA Artificial Sequence Description of ArtificialSequence Synthetic Sequence 40 gggaggacga ugcggaccua acucaaucaggaaccaaacc uagcacucuc auggccagac 60 gacucgcccg a 71 41 71 RNA ArtificialSequence Description of Artificial Sequence Synthetic Sequence 41gggaggacga ugcgggagau caacauuccu cuaguuuggu uccaaccuac acccccagac 60gacucgcccg a 71 42 71 RNA Artificial Sequence Description of ArtificialSequence Synthetic Sequence 42 gggaggacga ugcggaucuc gauccuucagcacuucauuu cauuccuuuc ugccccagac 60 gacucgcccg a 71 43 71 RNA ArtificialSequence Description of Artificial Sequence Synthetic Sequence 43gggaggacga ugcggacgau ccuuuccuua acauuucauc auuucucuug ugccccagac 60gacucgcccg a 71 44 71 RNA Artificial Sequence Description of ArtificialSequence Synthetic Sequence 44 gggaggacga ugcggugacg acaacucgacugcauaucuc acaacuccug ugccccagac 60 gacucgcccg a 71 45 72 RNA ArtificialSequence Description of Artificial Sequence Synthetic Sequence 45gggaggacga ugcggacuag accgcgaguc cauucaacuu gcccaaaaac cucccccaga 60cgacucgccc ga 72 46 70 RNA Artificial Sequence Description of ArtificialSequence Synthetic Sequence 46 gggaggacga ugcgggcgca ucgagcaacauccgauucgg auuccuccac ucccccagac 60 gacugcccga 70 47 50 RNA ArtificialSequence Description of Artificial Sequence Synthetic Sequence 47gggaggacga ugcggaacaa ugcacucguc gccguaaugg auguuuugcu 50 48 55 RNAArtificial Sequence Description of Artificial Sequence SyntheticSequence 48 gggaggacga ugcggaacaa ugcacucguc gccguaaugg auguuuugcu cccug55 49 55 RNA Artificial Sequence Description of Artificial SequenceSynthetic Sequence 49 gggaggacga ugcggaacaa ugcacucguc gccguaauggauguuuugcu cccug 55 50 55 RNA Artificial Sequence Description ofArtificial Sequence Synthetic Sequence 50 gggaggacga ugcggaacaaugcacucguc gccguaaugg auguuuugcu cccug 55 51 55 RNA Artificial SequenceDescription of Artificial Sequence Synthetic Sequence 51 gggaggacgaugcggaacaa ugcacucguc gccguaaugg auguuuugcu cccug 55 52 55 RNAArtificial Sequence Description of Artificial Sequence SyntheticSequence 52 gggaggacga ugcggaacaa ugcacucguc gccguaaugg auguuuugcu cccug55 53 55 RNA Artificial Sequence Description of Artificial SequenceSynthetic Sequence 53 gggaggacga ugcggaacaa ugcacucguc gccguaauggauguuuugcu cccug 55 54 55 RNA Artificial Sequence Description ofArtificial Sequence Synthetic Sequence 54 gggaggacga ugcggaacaaugcacucguc gccguaaugg auguuuugcu cccug 55 55 55 RNA Artificial SequenceDescription of Artificial Sequence Synthetic Sequence 55 gggaggacgaugcggaacaa ugcacucguc gccguaaugg auguuuugcu cccug 55 56 55 RNAArtificial Sequence Description of Artificial Sequence SyntheticSequence 56 gggaggacga ugcggaacaa ugcacucguc gccguaaugg auguuuugcu cccug55 57 55 RNA Artificial Sequence Description of Artificial SequenceSynthetic Sequence 57 gggaggacga ugcggaacaa ugcacucguc gccguaauggauguuuugcu cccug 55 58 55 RNA Artificial Sequence Description ofArtificial Sequence Synthetic Sequence 58 gggaggacga ugcggaacaaugcacucguc gccguaaugg auguuuugcu cccug 55 59 55 RNA Artificial SequenceDescription of Artificial Sequence Synthetic Sequence 59 gggaggacgaugcggaacaa ugcacucguc gccguaaugg auguuuugcu cccug 55 60 55 RNAArtificial Sequence Description of Artificial Sequence SyntheticSequence 60 gggaggacga ugcggaacaa ugcacucguc gccguaaugg auguuuugcu cccug55 61 55 RNA Artificial Sequence Description of Artificial SequenceSynthetic Sequence 61 gggaggacga ugcggaacaa ugcacucguc gccguaauggauguuuugcu cccug 55 62 55 RNA Artificial Sequence Description ofArtificial Sequence Synthetic Sequence 62 gggaggacga ugcggaacaaugcacucguc gccguaaugg auguuuugcu cccug 55 63 55 RNA Artificial SequenceDescription of Artificial Sequence Synthetic Sequence 63 gggaggacgaugcggaacaa ugcacucguc gccguaaugg auguuuugcu cccug 55 64 39 RNAArtificial Sequence Description of Artificial Sequence SyntheticSequence 64 gggaggacgn cgucgccgua auggauguuu ugcucccug 39 65 34 RNAArtificial Sequence Description of Artificial Sequence SyntheticSequence 65 gggaggacgn cgucgccgua auggauguuu ugcu 34

What is claimed is:
 1. A method for delivering a therapeutic agent to apatient having a disease in which tecnascin-C is expressed, comprising:covalently attaching a tenascin-C nucleic acid ligand to a therapeuticagent to form a complex, and administering said complex to said patient.2. The method of claim 1, wherein the disease in which tenascin-C isexpressed is selected from the group consisting of cancer,hyperproliferative skin diseases, and arthrosclerosis.
 3. The method ofclaim 2, wherein the cancer is selected from the group consisting oflung cancer, breast cancer, prostate cancer, colon cancer, astrocytomas,glioblastomas, melanomas, and sarcomas.
 4. The method of claim 1,wherein the disease in which tenascin-C is expressed is a disease inwhich tenascin-C is overexpressed.
 5. The method of claim 1, wherein theattachment of the tenascin-C nucleic acid ligand to a therapeutic agentis accomplished through the use of a linker.
 6. The method of claim 5,wherein said linker has the structure:


7. The method of claim 6, wherein the tenascin-C nucleic acid ligand issingle stranded.
 8. The method of claim 7, wherein the tenascin-Cnucleic acid ligand is RNA.
 9. The method of claim 8, wherein thetenascin-C nucleic acid ligand is comprised of 2′-fluoro (2′-F) modifiednucleotides.
 10. The method of claim 1, wherein the tenascin-C nucleicacid ligand is selected from the group consisting of SEQ ID NO:4-65.